Abstract The primary focus of my laboratory is the development of novel technologies for protein analysis, specifically centered around the concept of the proteoform. Proteoforms, each of which comprises a unique combination of amino acid sequence and post-translational modifications (PTMs), are the primary molecular effectors of cell function. Subtle sequence and PTM differences between proteoforms can completely alter their function and activity. We see comprehensive proteoform-level analysis of biological systems as absolutely essential to understanding their function, for both individual pathways and networks operative within cells, and more globally, to decipher the systems-biology-level dynamics and interactions that control cellular response. For example, one of our projects is to elucidate the interactome between specific nucleic acid sequences and the proteoforms bound to those DNA or RNA molecules. However, today's technology for global proteoform analysis in complex systems is in its infancy, offering both a great challenge and a great opportunity. We seek to develop novel strategies for comprehensive proteoform identification and quantification in complex systems. We envision combining information from multiple data streams, such as transcriptomic data (to reveal splice forms and genetic variation), bottom-up proteomics data (to reveal and localize PTMs), top-down proteomics data (to provide sequence tags for proteoform identifications), and intact mass measurements (to identify and quantify proteoforms, using information from all of the other data streams). Specific projects will develop the following: (1) robust tools for the construction of sample-specific proteoform databases; (2) new strategies for the discovery and localization of PTMs; (3) improved sample preparation, separation, and mass spectrometry methods for intact proteins; (4) synergistic approaches that utilize both intact mass measurements and selected top-down fragmentations to maximize proteoform identifications; and (5) visualization tools for proteoform families that show connections and changes between related proteoforms. We will integrate these methods and data streams together with powerful open-source software and accompanying protocols to make these capabilities widely available, enabling researchers everywhere to gain a deeper understanding of the functioning of their biological systems. We will apply our innovative tools to many cutting-edge projects with numerous collaborators, both because technology development is most meaningful in the context of relevant biological studies and because it will increase the adoption of proteoform analysis among scientists in the broader biomedical community.